Indirect Use of barley and wheat SSR primer sets146 barley210 wheat

Direct Development of oat-based SSR enriched librariesACATTATGCATC

USDA ARS Aberdeen, Molecular Genetics Laboratory Robert Campbell – Biological Technician Irene Shackelford – Biological Technician

EXPANSION OF OAT-BASED MICROSATELLITE RESOURCES USING BOTH DIRECT AND INDIRECT

STRATEGIESEric Jackson, J. Anderson, M. Carson, D. Obert, G. Hu, and M. Bonman

Genotype cfdF x cfd-O-303R_allele_1bmac387F x bmac387R_allele_2 (SSR)bmac387F x bmac387R_allele_3 (SSR)HvM52F x HvM52R_allele_1 (SSR)HvM52F x HvM52R_allele_2 (SSR)wmc389F x wmc389_O_r_allele_1 wmc389F x wmc389_O_r_allele_1 HvM62-T3-F x HvM62R_allele_1 HvM62F x HvM62-T1-R_allele_1 HvM62F x HvM62-O-R_allele_1 Bmag375F x Bmag_O_r_allele_1Bmag375F x Bmag_O_r_allele_2Ogle1040 190 -- 900 -- -- 180 620 120 -- 150 -- --TAM O-301 -- 300 -- 60 260 -- -- -- 230 155 290 160

Ogle157 -- 300 -- 60 -- 180 -- 120 -- 150 290 --Kanota 190 -- 900 -- 260 -- 620 120 230 150 290 --

A. maraccana wild type 190 -- -- -- -- -- -- -- -- 155 290 --A. maraccana domest. -- -- 900 60 -- -- -- -- -- 150 290 160

MN841801-1 (Rines, MN) 190 -- 900 60 -- 180 620 120 230 155 290 --Noble-2 190 300 900 -- -- 180 620 120 230 150 290 160

MN841801-1 (Chong, CA) NA 300 900 60 -- 180 -- 120 230 150 290 --Makuru 190 300 -- 60 -- 180 -- -- 230 150 290 --

TAM O-405 190 -- 900 -- -- -- 620 -- 230 150 -- 160Otana 190 -- 900 60 260 -- 620 120 -- 150 290 160

MN841810 -- -- 900 -- 260 -- -- 120 230 150 -- 160Stout 190 300 900 60 -- 180 620 120 230 150 290 160

SunII 190 300 -- -- 260 180 620 120 230 150 290 --

Polymorphic 3/6 3/6 3/6 5/6 4/6 3/6 3/6 3/6 3/6 2/6 3/6 2/6

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BARC83b (88bp -- TAM O-301)[(GTA)2(GCA)1(GTA)3(GCA)2(GTA)4] AAGCAAGGAA CGAGCAAGAG CAGTAGTAGC AGTAGTAGTA GCAGCAGTAG TAGTAGTATT CGTCATCTTC ATCGTCGTCG TAAATCCA BARC83b ( 82bp - - Og l e ) [ ( GTA) 2 ( GCA) 2 GTA) 2( GCAGCC) ( GTA) 3] AAGCAAGGAA CGAGC AAGAG CAGTAGTAGC AGCAGTAGTA GCAGCCGTAG TAGTA- - - - - C- TCATCTTC ATCGTCGTCG TAAATCCA BARC83a ( 73bp - - Og l e ) [ ( GTA) 1( GCA) 2( GTA) 4] AAGCAAGGAA CGAGCAAGAG CAGGTAGCAG CAGTAGTAGT AGTATCGTCA TCTTCATCGT CGTCGTAAAT CCA Bmag387 ( 231bp - - Og l e ) [ ( GA) ( GA) ( GCA) ( GA) ( GCA) ( GA) 4( GA) ( GA) ( GCA) ( GA) ] CGATGACCAT TGTATTGAAG CTCTAGGGGC CAATCTTATT TGGAGAGATG TGCAACCCCC TCAACAACTC MTACG CTGCC GCCTGCCACC GACGAGGCAC ATAGAAGCAG AGGAGAGATC CGGCGAGGTG GATTTGGTTA GCAAGATCCA CCCACCTACT CTTAGACAAA ACATGGCCAC ACGCCCTCAA GGCAAACCCT ACAACTACTG CTAACCACAC ATCAACATG A G

Do

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SSR Markers

Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAM Ogle/TAMAB_AM_17 AB_AM_19 AB_AM_20 AM_AB_28 AB_AM _29 AB_AM_31 AB_AM_32 AB_AM_34 AB_AM_35 AB_AM_37 AB_AM_53

100

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Fig. 2 Validation of candidate SSR primer sets designed from the AC - and ATG - libraries. Reactions were run with a 58 oC annealing temperature and were run out on 3% metaphor agarose in a lithium tetraborate (LB) buffer at 180V for 1.5h.

Og le/TAM Ogle/TAM Og le/TAM Og le/TAM Og le/TAM Ogle/TAM Ogle/TAM Og le/TAM Og le/TAM Og le/TAM Og le/TAMAB_AM_17 AB_AM_19 AB_AM_20 AM_AB_28 AB_AM _29 AB_AM_31 AB_AM_32 AB_AM_34 AB_AM_35 AB_AM_37 AB_AM_53

100

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Fig. 2 Validation of candidate SSR primer sets designed from the AC - and ATG - libraries. Reactions were run with a 58 oC annealing temperature and were run out on 3% metaphor agarose in a lithium tetraborate (LB) buffer at 180V for 1.5h.

Polymorphic markers:AB_AM_17 Dom.AB_AM_20 Dom.AB_AB_28 Co-dom.AB_AM_29 Dom.AB_AM_31 Dom.AB_AM_32 Dom.AB_AM_35 Co-dom.AB_AM_53 Dom.

AB_AM_17 >03_Oat A103 (848 -total) 160bp Ogle (TG)15

CCAAGAATTAATGCAAATTAGTCACTGTCCTAAAATCTATAAACTACAATCATTTTAAGGTTTTTTTTTGGCAAAGTGAAGATAATAAACGAAAATATCAGTTAACTAAAAAATGGAATATTATTTATAGACCAAAAGATCAGGGAGAGCAAGGTAGAAGTACTCGATGTTAAGAACAAATGTTTACTCAGCAAATGGATGTTT AAGTTACTAAACAAAAAGGGTATGTGGCAAGAGTTGCTGCAAAATAATAATTGTTCAATCGAGAAGACTTTGGCTC AGATGCGGAGTAAACCTAACA GACTCTCTATTCTAGAAAGGCCTCATGAAAGTTAAGGATGAAATTTTTAGCAAAGGTCTTTTCAATGTGTGTGTGTGTGTGTGTGTGTGTGTGTG GGGTGGTTACTACACTTCCATCTATGCACTCTAAGTCGTCGAA TTAAGCAACTTAAAATGAGGT ATACATTATAGAAAATATATCGTTGAAAACTTCAGATTTTATACTTTCTAATGGCATAATTTTTGTATTATATAGTTTAAATTATCTAGATCAAATTAATGACTTAAGTATACGCGCATGACTTATAAACCTGGATGGGAGGGAGTACATTAAACTGGTCCTTCATTTTAACNCTGCCCCAAAGTTGGGATNNTTTAATTGGAATCAAAAGAAATGGACCCGATTATCCGTCTAACATTAAAATACATTTTTGGTN CCNCCTTTTAAAGCCTTGCCATTGCCTGCANGGTCGANCTCTTAAAAGGATTCCCCCGGGGTANCCGAANCCCCGAANTTCCACTGGGCCCGNCGGTTTTNACAAACGTCCGTGGACTGGGGAAAANCCTGG

CONCULSIONS

31% that amplified between Ogle and TAM O-301 contained degenerative SSR motifs, while one complete SSR motif was identified.

78.6% sequenced from the AG- and ATG- libraries contained SSR motifs for which working primers could be designed.

25.5% of the markers were polymorphic between Ogle and TAM O-301 (agarose) and 43% of those were polymorphic.

Sequencing of the two libraries has yielded approximately 1700 candidate SSR primer sets. 1336 working primers 300 polymorphic in OT 130 co-dominant

Enough candidate SSR markers have been identified, so now the difficult task of validation and

mapping will begin.